Antithrombotic agents

ABSTRACT

This application relates to novel compounds of formula I (and their pharmaceutically acceptable salts), as defined herein, processes and intermediates for their preparation, pharmaceutical formulations comprising the novel compounds of formula I, and the use of the compounds of formula I as thrombin inhibitors.

This application claims the benefit of U.S. Provisional Application No.60/106,048, filed Oct. 28, 1998.

This invention relates to thrombin inhibitors which are usefulanticoagulants in mammals. In particular it relates to3-aminobenzo[b]thiophene derivatives having high anticoagulant activity,and antithrombotic activity. Thus, this invention relates to newinhibitors of thrombin, pharmaceutical compositions containing thecompounds as active ingredients, and the use of the compounds asanticoagulants for prophylaxis and treatment of pulmonary embolism,arterial thrombosis, in particular myocardial ischemia, myocardialinfarction and cerebral thrombosis, general hypercoagulable states andlocal hypercoagulable states, such as following angioplasty and coronarybypass operations, and generalized tissue injury as it relates to theinflammatory process. In addition, the antithrombotic agents are usefulas anticoagulants in in vitro applications.

The process of blood coagulation, thrombosis, is triggered by a complexproteolytic cascade leading to the formation of thrombin. Thrombinproteolytically removes activation peptides from the Aα-chains and theBβ-chains of fibrinogen, which is soluble in blood plasma, initiatinginsoluble fibrin formation.

Anticoagulation currently is achieved by the administration of heparinsand coumarins. Parenteral pharmacological control of coagulation andthrombosis is based on inhibition of thrombin through the use ofheparins. Heparins act indirectly on thrombin by accelerating theinhibitory effect of endogenous antithrombin III (the main physiologicalinhibitor of thrombin). Because antithrombin III levels vary in plasmaand because clot-bound thrombin seems resistant to this indirectmechanism, heparins can be an ineffective treatment. Because coagulationassays are believed to be associated with efficacy and with safety,heparin levels must be monitored with coagulation assays (particularlythe activated partial thromboplastin time (APTT) assay). Coumarinsimpede the generation of thrombin by blocking the posttranslationalgamma-carboxylation in the synthesis of prothrombin and other proteinsof this type. Because of their mechanism of action, the effect ofcoumarins can only develop slowly, 6-24 hours after administration.Further, they are not selective anticoagulants. Coumarins also requiremonitoring with coagulation assays (particularly the prothrombin time(PT) assay).

Antithrombotic diamines are disclosed in International PatentApplication Publication Number WO 97/25033.

Although the heparins and coumarins are effective anticoagulants, nocommercial drug has yet emerged from the promise for this class ofcompounds, there still exists a need for anticoagulants which actselectively on thrombin, and which, independent of antithrombin III,exert inhibitory action shortly after administration, preferably by anoral route, and do not interfere with lysis of blood clots, as requiredto maintain hemostasis.

The present invention is directed to the discovery that the compounds ofthe present invention, as defined below, are potent thrombin inhibitorsthat may have high bioavailability following oral administration.

According to the invention there is provided a compound of formula I (ora pharmaceutically acceptable salt thereof) ##STR1## wherein E is CR^(e)or N in which R^(e) is hydrogen, methyl, methoxy or halo;

R¹ is hydrogen or methyl;

R² is R^(2a) or R^(2b) in which

R^(2a) is --X² --(CH₂)_(n) --R^(f) in which X² is a direct bond,methylene or O; n is 1, 2 or 3; and R^(f) is 5-tetrazolyl, carboxy,[(1-4C)alkoxy]carbonyl or hydroxymethyl;

R^(2b) is --X² --(CH₂)_(m) --NR^(a) R^(b) in which X² is a direct bond,methylene or O; m is 1, 2 or 3; provided that when m is 1, then X² is adirect bond; and R^(a) and R^(b) are independently hydrogen or(1-3C)alkyl or the group NR^(a) R^(b) is pyrrolidino, piperidino ormorpholino;

R³ is --X3--(CH₂)_(s) --NR^(s) R^(t) in which X³ is a direct bond,methylene or O; s is 1 or 2; provided that when s is 1, then X³ is adirect bond; and R^(s) and R^(t) are independently hydrogen or(1-3C)alkyl or the group NR^(s) R^(t) is pyrrolidino, piperidino ormorpholino; and

R⁶ is hydrogen, hydroxy or methoxy.

In this specification, the following definitions are used, unlessotherwise described: Halo is fluoro, chloro, bromo or iodo. Alkyl,alkoxy, etc. denote both straight and branched groups; but reference toan individual radical such as "propyl" embraces only the straight chain("normal") radical, a branched chain isomer such as "isopropyl" beingspecifically denoted.

It will be appreciated that certain compounds of formula I (or salts orprodrugs, etc.) may exist in, and be isolated in, isomeric forms,including cis- or trans-isomers, as well as optically active, racemic,or diastereomeric forms. It is to be understood that the presentinvention encompasses a compound of formula I as a mixture ofdiastereomers, as well as in the form of an individual diastereomer, andthat the present invention encompasses a compound of formula I as amixture of enantiomers, as well as in the form of an individualenantiomer, any of which mixtures or form possesses inhibitoryproperties against thrombin, it being well known in the art how toprepare or isolate particular forms and how to determine inhibitoryproperties against thrombin by standard tests including those describedbelow.

In addition, a compound of formula I (or salt of prodrug, etc.) mayexhibit polymorphism or may form a solvate with water or an organicsolvent. The present invention also encompasses any such polymorphicform, any solvate or any mixture thereof.

Particular values are listed below for radicals, substituents, andranges, for illustration only, and they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents.

A particular value for a (1-3C)alkyl group is, for example, methyl,ethyl, propyl or isopropyl, and for a (1-4C)alkoxy group is, forexample, methoxy, ethoxy, isopropoxy or t-butoxy.

A particular value, independently, for E is CR^(e) in which R^(e) ishydrogen or methoxy; for R¹ is hydrogen or methyl; for R² is R^(2a) orR^(2b) in which R^(2a) is --X² --(CH₂)_(n) --R^(f) in which X² is O; nis 2; and R^(f) is carboxy or methoxycarbonyl; R^(2b) is --X²--(CH₂)_(m) --NR^(a) R^(b) in which X² is O; m is 2; and the groupNR^(a) R^(b) is pyrrolidino; for R³ is --X³ --(CH₂)_(s) --NR^(s) R^(t)in which X³ is a direct bond or O, s is 1 or 2 (provided that when s is1, then X³ is a direct bond), and the group NR^(s) R^(t) is pyrrolidino;and for R⁶ is hydrogen or hydroxy.

A more particular value for R² is R^(2a). A further particular value forR² is R^(2b).

A particular compound of formula I is one in which E is CR^(e) in whichR^(e) is methoxy and R³ is pyrrolidinomethyl.

Specific compounds of formula I are described in the accompanyingExamples. The compound described as Example 6 (or a pharmaceuticallyacceptable salt thereof) is a preferred species.

A pharmaceutically acceptable salt of an antithrombotic agent of theinstant invention includes one which is an acid-addition salt made withan acid which provides a pharmaceutically acceptable anion. Thus, anacid addition salt of a novel compound of formula I as provided abovemade with an acid which affords a pharmaceutically acceptable anionprovides a particular aspect of the invention. Examples of such acidsare provided hereinbelow. In addition, a compound of formula I whichcontains an acidic moiety forms a salt made with a base which provides apharmaceutically acceptable anion.

As an additional aspect of the invention there is provided apharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a compound offormula I (or a pharmaceutically acceptable salt thereof) as provided inany of the above descriptions.

A compound of formula I may be made by processes which include processesknown in the chemical art for the production of compounds structurallyrelated to a compound of formula I or by a novel process describedherein. A process for a compound of formula I (or a pharmaceuticallyacceptable salt thereof), novel processes for a compound of formula Iand novel intermediates for the manufacture of a compound of formula Ias defined above provide further features of the invention and areillustrated by the following procedures in which the meanings of thegeneric radicals are as defined above, unless otherwise specified. Itwill be recognized that it may be preferred or necessary to prepare acompound of formula I in which a functional group is protected using aconventional protecting group, then to remove the protecting group toprovide the compound of formula I.

In general, a compound of formula I may be prepared according to one ofthe routes outlined in Scheme I, and which are described in theexamples, in which each of Q¹, Q², Q³ and Q⁶, respectively, represents avalue defined for the groups R¹, R², R³ and R⁶, a protected version ofsuch a group, or moiety which can be further elaborated into such agroup; and M(+) denotes a suitable counterion for the deprotonatedaniline of formula (A). Final conversion of a group Q¹, Q², Q³ or Q⁶into R¹, R², R³, or R⁶ is carried out at a convenient point, consistentwith the chemistry employed. Conveniently, the species of formula (A) isprepared by deprotonating the corresponding aniline using a strong baseand M(+) denotes a cation derived from an alkali metal, for examplelithium, sodium or potassium. It will be recognized that a number ofother routes may be used, particularly those involving displacement ofthe bromide from a 3-bromobenzo[b]thiophene analogous to that of formula(B), but in which the sulfur is not oxidized. ##STR2##

Thus, there is provided a process for preparing a compound of formula I(or a pharmaceutically acceptable salt thereof) as provided in any ofthe above descriptions which is selected from:

(a) for a compound of formula I in which R² is --X² --(CH₂)_(n) --R^(f)or --X² --(CH₂)_(m) --NR^(a) R^(b) where X² is O, alkylating acorresponding phenol of formula II ##STR3## using a compound of formulaL-(CH₂)_(n) --R^(f) or L-(CH₂)_(m) --NR^(a) R^(b) (or a protectedderivative thereof), wherein L denotes a conventional leaving group,using a standard alkylating procedure;

(b) for a compound of formula I in which R^(f) is carboxy, decomposingthe ester of a corresponding compound of formula I in which R^(f) is[(1-4C)alkoxy]carbonyl; and

(c) deoxygenating the sulfoxide of a corresponding compound of formulaIII ##STR4## whereafter, for any of the above procedures, when afunctional group is protected using a protecting group, removing theprotecting group; and

whereafter, for any of the above procedures, when a pharmaceuticallyacceptable salt of a compound of formula I is required, it is obtainedby reacting the basic form of such a compound of formula I with an acidaffording a physiologically acceptable counterion, or, for a compound offormula I which bears an acidic moiety, reacting the acidic form of sucha compound of formula I with a base which affords a pharmaceuticallyacceptable cation, or by any other conventional procedure;

and wherein, unless otherwise described, R¹, R², R³ and R⁶ have thevalues described above.

As used herein, a leaving group is a moiety which is displaced in anucleophilic substitution reaction, for example a halo group (such aschloro, bromo or iodo), a sulfonate ester group (such asmethylsulfonyloxy, p-toluylsulfonyloxy or trifluoromethylsulfonyloxy),or the reactive species derived from treating an alcohol withtriphenylphospine, diethyl azodicarboxylate and triethyl amine (in aMitsunobu reaction). Deoxygenation of the sulfoxide may be performed inany conventional manner, provided the reagent used does not reduceanother functionality within the molecule.

Novel intermediate or starting material compounds, such as a novelphenol of formula II or sulfoxide of formula III, provide furtheraspects of the invention.

As mentioned above, a compound corresponding to a compound of formula Ibut in which a functional group is protected may serve as anintermediate for a compound of formula I. Accordingly, such protectedintermediates for a novel compound of formula I provide further aspectsof the invention. Thus, as one particular aspect of the invention, thereis provided a compound corresponding to a novel compound of formula I asdefined above in which R⁶ which is hydroxy, but in which thecorresponding substituent is --ORP in place of hydroxy, wherein RP is aphenol protecting group other than methyl. Phenol protecting groups arewell known in the art, for example as described in T. W. Greene and P.G. M. Wuts, "Protecting Groups in Organic Synthesis" (1991). Particularvalues of RP include, for example, isopropyl. Further, RP may denote afunctionalized resin, for example as disclosed in H. V. Meyers, et al.,Molecular Diversity, (1995), 1, 13-20.

As mentioned above, the invention includes pharmaceutically acceptablesalts of the thrombin inhibiting compounds defined by the above formulaI. A compound of formula I which bears an acidic moiety forms salts withpharmaceutically acceptable bases. Such a pharmaceutically acceptablesalt may be made with a base which affords a pharmaceutically acceptablecation, which includes alkali metal salts (especially sodium andpotassium), alkaline earth metal salts (especially calcium andmagnesium), aluminum salts and ammonium salts, as well as salts madefrom physiologically acceptable organic bases such as triethylamine,morpholine, piperidine and triethanolamine. The potassium and sodiumsalt forms are particularly preferred.

A particular compound of formula I which possesses one or moresufficiently basic functional groups to react with any of a number ofinorganic and organic acids affording a physiologically acceptablecounterion forms a pharmaceutically acceptable acid addition salt. Acidscommonly employed to form pharmaceutically acceptable acid additionsalts are inorganic acids such as hydrochloric acid, hydrobromic acid,hydroiodic acid, sulfuric acid, phosphoric acid, and the like, andorganic acids such as p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid, and the like. Examples of suchpharmaceutically acceptable salts thus are the sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate,dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide,iodide, acetate, propionate, decanoate, caprylate, acrylate, formate,isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate,succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycollate, tartrate,methanesulfonate, propanesulfonate, naphthalene-1-sulfonate,naphthalene-2-sulfonate, mandelate, and the like. Preferredpharmaceutically acceptable acid addition salts include those formedwith mineral acids such as hydrochloric acid, hydrobromic acid andsulfuric acid.

If not commercially available, the necessary starting materials for thepreparation of a compound of formula I may be prepared by procedureswhich are selected from standard techniques of organic chemistry,including aromatic and heteroaromatic substitution and transformation,from techniques which are analogous to the syntheses of known,structurally similar compounds, and techniques which are analogous tothe above described procedures or procedures described in the Examples.It will be clear to one skilled in the art that a variety of sequencesis available for the preparation of the starting materials. Startingmaterials which are novel provide another aspect of the invention.

Selective methods of protection and deprotection are well known in theart for preparation of compounds such as those corresponding to acompound of formula I but in which R⁶ is ORP discussed above and aredescribed in the examples.

Generally, the compounds of the invention are isolated best in the formof acid addition salts. Salts of the compounds of formula I formed withacids such as those mentioned above are useful as pharmaceuticallyacceptable salts for administration of the antithrombotic agents and forpreparation of formulations of these agents. Other acid addition saltsmay be prepared and used in the isolation and purification of thecompounds.

As noted above, the optically active isomers and diastereomers of thecompounds of formula I are also considered part of this invention. Suchoptically active isomers may be prepared from their respective opticallyactive precursors by the procedures described above, or by resolving theracemic mixtures. This resolution can be carried out by derivatizationwith a chiral reagent followed by chromatography or by repeatedcrystallization. Removal of the chiral auxiliary by standard methodsaffords substantially optically pure isomers of the compounds of thepresent invention or their precursors. Further details regardingresolutions can be obtained in Jacques, et al., Enantiomers, Racemates,and Resolutions, John Wiley & Sons, 1981.

The compounds of the invention are believed to selectively inhibitthrombin over other proteinases and nonenzyme proteins involved in bloodcoagulation without appreciable interference with the body's naturalclot lysing ability (the compounds have a low inhibitory effect onfibrinolysis). Further, such selectivity is believed to permit use withthrombolytic agents without substantial interference with thrombolysisand fibrinolysis.

The invention provides a method of inhibiting thrombin comprising usingan effective amount of a compound of formula I (or a pharmaceuticallyacceptable salt thereof) as provided in any of the descriptions herein.

The invention in one of its aspects provides a method of inhibitingthrombin in a mammal comprising administering to a mammal in need oftreatment an effective (thrombin inhibiting) dose of a compound offormula I.

In another of its aspects, the invention provides a method of treating athromboembolic disorder comprising administering to a mammal in need oftreatment an effective (thromboembolic disorder therapeutic and/orprophylactic amount) dose of a compound of formula I.

The invention in another of its aspects provides a method of inhibitingcoagulation in mammals comprising administering to a mammal in need oftreatment an effective (coagulation inhibiting) dose of a compound offormula I.

The thrombin inhibition, coagulation inhibition and thromboembolicdisorder treatment contemplated by the present method includes bothmedical therapeutic and/or prophylactic treatment as appropriate.

In a further embodiment the invention relates to treatment, in a humanor animal, of conditions where inhibition of thrombin is required. Thecompounds of the invention are expected to be useful in animals,including man, in treatment or prophylaxis of thrombosis andhypercoagulability in blood and tissues. Disorders in which thecompounds have a potential utility are in treatment or prophylaxis ofthrombosis and hypercoagulability in blood and tissues. Disorders inwhich the compounds have a potential utility, in treatment and/orprophylaxis, include venous thrombosis and pulmonary embolism, arterialthrombosis, such as in myocardial ischemia, myocardial infarction,unstable angina, thrombosis-based stroke and peripheral arterialthrombosis. Further, the compounds have expected utility in thetreatment or prophylaxis of atherosclerotic disorders (diseases) such ascoronary arterial disease, cerebral arterial disease and peripheralarterial disease. Further, the compounds are expected to be usefultogether with thrombolytics in myocardial infarction. Further, thecompounds have expected utility in prophylaxis for reocclusion afterthrombolysis, percutaneous transluminal angioplasty (PTCA) and coronarybypass operations. Further, the compounds have expected utility inprevention of rethrombosis after microsurgery. Further, the compoundsare expected to be useful in anticoagulant treatment in connection withartificial organs and cardiac valves. Further, the compounds haveexpected utility in anticoagulant treatment in hemodialysis anddisseminated intravascular coagulation. A further expected utility is inrinsing of catheters and mechanical devices used in patients in vivo,and as an anticoagulant for preservation of blood, plasma and otherblood products in vitro. Still further, the compounds have expectedutility in other diseases where blood coagulation could be a fundamentalcontributing process or a source of secondary pathology, such as cancer,including metastasis, inflammatory diseases, including arthritis, anddiabetes. The anti-coagulant compound is administered orally,parenterally e.g. by intravenous infusion (iv), intramuscular injection(im) or subcutaneously (sc).

The specific dose of a compound administered according to this inventionto obtain therapeutic and/or prophylactic effects will, of course, bedetermined by the particular circumstances surrounding the case,including, for example, the compound administered, the rate ofadministration, the route of administration, and the condition beingtreated.

A typical daily dose for each of the above utilities is between about0.01 mg/kg and about 1000 mg/kg. The dose regimen may vary e.g. forprophylactic use a single daily dose may be administered or multipledoses such as 3 or 5 times daily may be appropriate. In critical caresituations a compound of the invention is administered by iv infusion ata rate between about 0.01 mg/kg/h and about 20 mg/kg/h and preferablybetween about 0.1 mg/kg/h and about 5 mg/kg/h.

The method of this invention also is practiced in conjunction with aclot lysing agent e.g. tissue plasminogen activator (t-PA), modifiedt-PA, streptokinase or urokinase. In cases when clot formation hasoccurred and an artery or vein is blocked, either partially or totally,a clot lysing agent is usually employed. A compound of the invention canbe administered prior to or along with the lysing agent or subsequent toits use, and preferably further is administered along with aspirin toprevent the reoccurrence of clot formation.

The method of this invention is also practiced in conjunction with aplatelet glycoprotein receptor (IIb/IIIa) antagonist, that inhibitsplatelet aggregation. A compound of the invention can be administeredprior to or along with the IIb/IIIa antagonist or subsequent to its useto prevent the occurrence or reoccurrence of clot formation.

The method of this invention is also practiced in conjunction withaspirin. A compound of the invention can be administered prior to oralong with aspirin or subsequent to its use to prevent the occurrence orreoccurrence of clot formation. As stated above, preferably a compoundof the present invention is administered in conjunction with a clotlysing agent and aspirin.

This invention also provides pharmaceutical formulations for use in theabove described therapeutic method. Pharmaceutical formulations of theinvention comprise an effective thrombin inhibiting amount of a compoundof formula I in association with a pharmaceutically acceptable carrier,excipient or diluent. For oral administration the antithromboticcompound is formulated in gelatin capsules or tablets which may containexcipients such as binders, lubricants, disintegration agents and thelike. For parenteral administration the antithrombotic is formulated ina pharmaceutically acceptable diluent e.g. physiological saline (0.9percent), 5 percent dextrose, Ringer's solution and the like.

The compound of the present invention can be formulated in unit dosageformulations comprising a dose between about 0.1 mg and about 1000 mg.Preferably the compound is in the form of a pharmaceutically acceptablesalt such as for example the sulfate salt, acetate salt or a phosphatesalt. An example of a unit dosage formulation comprises 5 mg of acompound of the present invention as a pharmaceutically acceptable saltin a 10 mL sterile glass ampoule. Another example of a unit dosageformulation comprises about 10 mg of a compound of the present inventionas a pharmaceutically acceptable salt in 20 mL of isotonic salinecontained in a sterile ampoule.

The compounds can be administered by a variety of routes including oral,rectal, transdermal, subcutaneous, intravenous, intramuscular, andintranasal. The compounds of the present invention are preferablyformulated prior to administration. Another embodiment of the presentinvention is a pharmaceutical formulation comprising an effective amountof a novel compound of formula I or a pharmaceutically acceptable saltor solvate thereof in association with a pharmaceutically acceptablecarrier, diluent or excipient therefor.

The active ingredient in such formulations comprises from 0.1 percent to99.9 percent by weight of the formulation. By "pharmaceuticallyacceptable" it is meant the carrier, diluent or excipient must becompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof.

The present pharmaceutical formulations are prepared by known proceduresusing well known and readily available ingredients. The compositions ofthis invention may be formulated so as to provide quick, sustained, ordelayed release of the active ingredient after administration to thepatient by employing procedures well known in the art. In making thecompositions of the present invention, the active ingredient willusually be admixed with a carrier, or diluted by a carrier, or enclosedwithin a carrier which may be in the form of a capsule, sachet, paper orother container. When the carrier serves as a diluent, it may be asolid, semi-solid or liquid material which acts as a vehicle, excipientor medium for the active ingredient. Thus, the compositions can be inthe form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols, (as asolid or in a liquid medium), soft and hard gelatin capsules,suppositories, sterile injectable solutions, sterile packaged powders,and the like.

The following formulation examples are illustrative only and are notintended to limit the scope of the invention in any way. "Activeingredient," of course, means a compound according to Formula I or apharmaceutically acceptable salt or solvate thereof.

Formulation 1

Hard gelatin capsules are prepared using the following ingredients:

    ______________________________________                                                            Quantity                                                    (mg/capsule)                                                                ______________________________________                                        Active ingredient     250                                                       Starch, dried 200                                                             Magnesium stearate 10                                                         Total 460 mg                                                                ______________________________________                                    

Formulation 2

A tablet is prepared using the ingredients below:

    ______________________________________                                                             Quantity                                                   (mg/tablet)                                                                 ______________________________________                                        Active ingredient      250                                                      Cellulose, microcrystalline 400                                               Silicon dioxide, fumed 10                                                     Stearic acid 5                                                                Total 665 mg                                                                ______________________________________                                    

The components are blended and compressed to form tablets each weighing665 mg.

Formulation 3

An aerosol solution is prepared containing the following components:

    ______________________________________                                                            Weight                                                    ______________________________________                                        Active ingredient     0.25                                                      Ethanol 25.75                                                                 Propellant 22 (Chlorodifluoromethane) 70.00                                   Total 100.00                                                                ______________________________________                                    

The active compound is mixed with ethanol and the mixture added to aportion of the propellant 22, cooled to -30° C. and transferred to afilling device. The required amount is then fed to a stainless steelcontainer and diluted with the remainder of the propellant. The valveunits are then fitted to the container.

Formulation 4

Tablets, each containing 60 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient        60     mg                                              Starch 45 mg                                                                  Microcrystalline cellulose 35 mg                                              Polyvinylpyrrolidone (as 10% solution in 4 mg                                 water)                                                                        Sodium carboxymethyl starch 4.5 mg                                            Magnesium stearate 0.5 mg                                                     Talc 1 mg                                                                     Total 150 mg                                                                ______________________________________                                    

The active ingredient, starch and cellulose are passed through a No. 45mesh U.S. sieve and mixed thoroughly. The aqueous solution containingpolyvinylpyrrolidone is mixed with the resultant powder, and the mixturethen is passed through a No. 14 mesh U.S. sieve. The granules soproduced are dried at 50° C. and passed through a No. 18 mesh U.S.Sieve. The sodium carboxymethyl starch, magnesium stearate and talc,previously passed through a No. 60 mesh U.S. sieve, are then added tothe granules which, after mixing, are compressed on a tablet machine toyield tablets each weighing 150 mg.

Formulation 5

Capsules, each containing 80 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient  80 mg                                                        Starch 59 mg                                                                  Microcrystalline cellulose 59 mg                                              Magnesium stearate  2 mg                                                      Total 200 mg                                                                ______________________________________                                    

The active ingredient, cellulose, starch, and magnesium stearate areblended, passed through a No. 45 mesh U.S. sieve, and filled into hardgelatin capsules in 200 mg quantities.

Formulation 6

Suppositories, each containing 225 mg of active ingredient, are made asfollows:

    ______________________________________                                        Active ingredient    225 mg                                                     Saturated fatty acid glycerides 2,000 mg                                      Total 2,225 mg                                                              ______________________________________                                    

The active ingredient is passed through a No. 60 mesh U.S. sieve andsuspended in the saturated fatty acid glycerides previously melted usingthe minimum heat necessary. The mixture is then poured into asuppository mold of nominal 2 g capacity and allowed to cool.

Formulation 7

Suspensions, each containing 50 mg of active ingredient per 5 ml dose,are made as follows:

    ______________________________________                                        Active ingredient       50     mg                                               Sodium carboxymethyl cellulose 50 mg                                          Syrup 1.25 mL                                                                 Benzoic acid solution 0.10 mL                                                 Flavor q.v.                                                                   Color q.v.                                                                    Purified water to total 5 mL                                                ______________________________________                                    

The active ingredient is passed through a No. 45 mesh U.S. sieve andmixed with the sodium carboxymethyl cellulose and syrup to form a smoothpaste. The benzoic acid solution, flavor and color are diluted with aportion of the water and added, with stirring. Sufficient water is thenadded to produce the required volume.

Formulation 8

An intravenous formulation may be prepared as follows:

    ______________________________________                                        Active ingredient     100    mg                                                 Isotonic saline 1,000 mL                                                    ______________________________________                                    

The solution of the above ingredients generally is administeredintravenously to a subject at a rate of 1 mL per minute.

The ability of the compounds of the present invention to be an effectiveand orally active thrombin inhibitor are evaluated in one or more of thefollowing assays.

The compounds provided by the invention (formula I) selectively inhibitthe action of thrombin in mammals. The inhibition of thrombin isdemonstrated by in vitro inhibition of the amidase activity of thrombinas measured in an assay in which thrombin hydrolyzes the chromogenicsubstrate, N-benzoyl-L-phenylalanyl-L-valyl-L-arginyl-p-nitroanilide,N-benzoyl-L-Phe-L-Val-L-Arg-p-nitroanilide.

The assay is carried out by mixing 50 μL buffer (0.03 M Tris, 0.15 MNaCl, pH 7.4) with 25 μL of human thrombin solution (purified humanthrombin, Enzyme Research Laboratories, South Bend, Ind., at 8 NIHunits/mL) and 25 μL of test compound in a solvent (50% aqueous methanol(v:v)). Then 150 μL of an aqueous solution of the chromogenic substate(at 0.25 mg/mL) are added and the rates of hydrolysis of the substrateare measured by monitoring the reactions at 405 nm for the release ofp-nitroaniline. Standard curves are constructed by plotting freethrombin concentration against hydrolysis rate. The hydrolysis ratesobserved with test compounds are then converted to "free thrombin"values in the respective assays by use of the standard curves. The boundthrombin (bound to test compound) is calculated by subtracting theamount of free thrombin observed in each assay from the known initialamount of thrombin used in the assay. The amount of free inhibitor ineach assay is calculated by subtracting the number of moles of boundthrombin from the number of moles of added inhibitor (test compound).

The Kass value is the hypothetical equilibrium constant for the reactionbetween thrombin and the test compound (I). ##EQU1##

Kass is calculated for a range of concentrations of test compounds andthe mean value reported in units of liter per mole. In general, athrombin inhibiting compound of formula I of the instant inventionexhibits a Kass of 0.1×10⁶ L/mole or much greater.

By substantially following the procedures described above for humanthrombin, and using other human blood coagulation system serineproteases and using fibrinolytic system serine proteases, with theappropriate chromogenic substrates, identified below, the selectivity ofthe compounds of the present invention with respect to the coagulationfactor serine proteases and to the fibronolytic serine proteases areevaluated as well as their substantial lack of interference with humanplasma clot fibrinolysis.

Human factors X, Xa, IXa, XIa, and XIIa are purchased from EnzymeResearch Laboratories, South Bend, Ind.; human urokinase from LeoPharmaceuticals, Denmark; and recombinant activated Protein C (aPC) isprepared at Eli Lilly and Co. substantially according to U.S. Pat. No.4,981,952. Chromogenic substrates:N-Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (for factor Xa);N-Cbz-D-Arg-Gly-Arg-p-nitroanilide (for factor IXa assay as the factorXa substrate); Pyroglutamyl-Pro-Arg-p-nitroanilide (for Factor XIa andfor aPC); H-D-Pro-Phe-Arg-p-nitroanilide (for factor XIIa); andPyroglutamyl-Gly-Arg-p-nitroanilide (for urokinase); are purchased fromKabi Vitrum, Stockholm, Sweden, or from Midwest Biotech, Fishers, Ind.Bovine trypsin is purchased from Worthington Biochemicals, Freehold,N.J., and human plasma kallikrein from Kabi Vitrum, Stockholm, Sweden.Chromogenic substrate H-D-Pro-Phe-Arg-p-nitroanilide for plasmakallikrein is purchased from Kabi Vitrum, Stockholm, Sweden.N-Benzoyl-Phe-Val-Arg-p-nitroanilide, the substrate for human thrombinand for trypsin, is synthesized according to procedures described abovefor the compounds of the present invention, using known methods ofpeptide coupling from commercially available reactants, or purchasedfrom Midwest Biotech, Fishers, Ind.

Human plasmin is purchased from Boehringer Mannheim, Indianapolis, Ind.;nt-PA is purchased as single chain activity reference from AmericanDiagnostica, Greenwich, Connecticut; modified-t-PA6 (mt-PA6) is preparedat Eli Lilly and Company by procedure known in the art (See, Burck, etal., J. Biol. Chem., 265, 5120-5177 (1990). Plasmin chromogenicsubstrate H-D-Val-Leu-Lys-p-nitroanilide and tissue plasminogenactivator (t-PA) substrate H-D-Ile-Pro-Arg-p-nitroanilide are purchasedfrom Kabi Vitrum, Stockholm, Sweden.

In the chromogenic substrates described above the three-letter symbolsIle, Glu, Gly, Pro, Arg, Phe, Val, Leu and Lys are used to indicate thecorresponding amino acid group isoleucine, glutamic acid, glycine,proline, arginine, phenylalanine, valine, leucine and lysine,respectively.

Thrombin inhibitors preferably should spare fibrinolysis induced byurokinase, tissue plasminogen activator (t-PA) and steptokinase. Thiswould be important to the therapeutic use of such agents as an adjunctto streptokinase, t-PA or urokinase thrombolytic therapy and to the useof such agents as an endogenous fibrinolysis-sparing (with respect tot-PA and urokinase) antithrombotic agents. In addition to the lack ofinterference with the amidase activity of the fibrinolytic proteases,such fibrinolytic system sparing can be studied by the use of humanplasma clots and their lysis by the respective fibrinolytic plasminogenactivators.

Materials

Dog plasma is obtained from conscious mixed-breed hounds (either sexButler Farms, Clyde, N.Y., U.S.A.) by venipuncture into 3.8 percentcitrate. Fibrinogen is prepared from fresh dog plasma and humanfibrinogen is prepared from in-date ACD human blood at the fraction I-2according to previous procedures and specifications. Smith, Biochem. J.,185, 1-11 (1980); and Smith, et al., Biochemistry, 11, 2958-2967,(1972). Human fibrinogen (98 percent pure/plasmin free) is from AmericanDiagnostica, Greenwich, Connecticut. Radiolabeling of fibrinogen I-2preparations is performed as previously reported. Smith, et al.,Biochemistry, 11, 2958-2967, (1972). Urokinase is purchased from LeoPharmaceuticals, Denmark, as 2200 Ploug units/vial. Streptokinase ispurchased from Hoechst-Roussel Pharmaceuticals, Somerville, N.J.

Methods--Effects on Lysis of Human Plasma Clots by t-PA

Human plasma clots are formed in micro test tubes by adding 50 μLthrombin (73 NIH unit/mL) to 100 μL human plasma which contains 0.0229μCi 125-iodine labeled fibrinogen. Clot lysis is studied by overlayingthe clots with 50 μL of urokinase or streptokinase (50, 100, or 1000unit/mL) and incubating for 20 hours at room temperature. Afterincubation the tubes are centrifuged in a Beckman Microfuge. 25 μL ofsupernate is added into 1.0 mL volume of 0.03 M tris/0.15 M NaCl bufferfor gamma counting. Counting controls 100 percent lysis are obtained byomitting thrombin (and substituting buffer). The thrombin inhibitors areevaluated for possible interference with fibrinolysis by including thecompounds in the overlay solutions at 1, 5, and 10 μg/mL concentrations.Rough approximations of IC₅₀ values are estimated by linearextrapolations from data points to a value which would represent 50percent of lysis for that particular concentration of fibrinolyticagent.

Anticoagulant Activity

Materials

Dog plasma and rat plasma are obtained from conscious mixed-breed hounds(either sex, Butler Farms, Clyde, N.Y., U.S.A.) or from anesthetizedmale Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis,Ind., U.S.A.) by venipuncture into 3.8 percent citrate. Fibrinogen isprepared from in-date ACD human blood as the fraction I-2 according toprevious procedures and specifications. Smith, Biochem. J., 185, 1-11(1980); and Smith, et al., Biochemistry, 11, 2958-2967 (1972). Humanfibrinogen is also purchased as 98 percent pure/plasmin free fromAmerican Diagnostica, Greenwich, Conn. Coagulation reagents Actin,Thromboplastin, Innovin and Human plasma are from Baxter HealthcareCorp., Dade Division, Miami, Fla. Bovine thrombin from Parke-Davis(Detroit, Mich.) is used for coagulation assays in plasma.

Methods

Anticoagulation Determinations

Coagulation assay procedures are as previously described. Smith, et al.,Thrombosis Research, 50, 163-174 (1988). A CoAScreener coagulationinstrument (American LABor, Inc.) is used for all coagulation assaymeasurements. The prothrombin time (PT) is measured by adding 0.05 mLsaline and 0.05 mL Thromboplastin-C reagent or recombinant human tissuefactor reagent (Innovin) to 0.05 mL test plasma. The activated partialthromboplastin time (APTT) is measured by incubation of 0.05 mL testplasma with 0.05 mL Actin reagent for 120 seconds followed by 0.05 mLCaCl₂ (0.02 M). The thrombin time (TT) is measured by adding 0.05 mLsaline and 0.05 mL thrombin (10 NIH units/mL) to 0.05 mL test plasma.The compounds of formula I are added to human or animal plasma over awide range of concentrations to determine prolongation effects on theAPTT, PT, and TT assays. Linear extrapolations are performed to estimatethe concentrations required to double the clotting time for each assay.

Animals

Male Sprague Dawley rats (350-425 gm, Harlan Sprague Dawley Inc.,Indianapolis, Ind.) are anesthetized with xylazine (20 mg/kg, s.c.) andketamine (120 mg/kg, s.c.) and maintained on a heated water blanket (37°C.). The jugular vein(s) is cannulated to allow for infusions.

Arterio-Venous shunt model

The left jugular vein and right carotid artery are cannulated with 20 cmlengths of polyethylene PE 60 tubing. A 6 cm center section of largertubing (PE 190) with a cotton thread (5 cm) in the lumen, is frictionfitted between the longer sections to complete the arterio-venous shuntcircuit. Blood is circulated through the shunt for 15 min before thethread is carefully removed and weighed. The weight of a wet thread issubtracted from the total weight of the thread and thrombus (see J. R.Smith, Br J Pharmacol, 77:29, 1982). In this model preferred compoundsof the instant invention reduce the net clot weight to approximately25-30% of control, or even lower, at an i.v. dose of 33.176 μmol/kg/h.

FeCl₃ model of arterial injury

The carotid arteries are isolated via a midline ventral cervicalincision. A thermocouple is placed under each artery and vesseltemperature is recorded continuously on a strip chart recorder. A cuffof tubing (0.058 ID×0.077 OD×4 mm, Baxter Med. Grade Silicone), cutlongitudinally, is placed around each carotid directly above thethermocouple. FeCl₃ hexahydrate is dissolved in water and theconcentration (20 percent) is expressed in terms of the actual weight ofFeCl₃ only. To injure the artery and induce thrombosis, 2.85 μL ispipetted into the cuff to bathe the artery above the thermocouple probe.Arterial occlusion is indicated by a rapid drop in temperature. The timeto occlusion is reported in minutes and represents the elapsed timebetween application of FeCl₃ and the rapid drop in vessel temperature(see K. D. Kurz, Thromb. Res., 60:269, 1990).

Spontaneous thrombolysis model

In vitro data suggests that thrombin inhibitors inhibit thrombin and, athigher concentrations, may inhibit other serine proteases, such asplasmin and tissue plasminogen activator. To assess if the compoundsinhibit fibrinolysis in vivo, the rate of spontaneous thrombolysis isdetermined by implanting a labeled whole blood clot into the pulmonarycirculation. Rat blood (1 mL) is mixed rapidly with bovine thrombin (4IU, Parke Davis) and ¹²⁵ I human Fibrogen (5 μCi, ICN), immediatelydrawn into silastic tubing and incubated at 37° C. for 1 hour. The agedthrombus is expelled from the tubing, cut into 1 cm segments, washed 3×in normal saline and each segment is counted in a gamma counter. Asegment with known counts is aspirated into a catheter that issubsequently implanted into the jugular vein. The catheter tip isadvanced to the vicinity of the right atrium and the clot is expelled tofloat into the pulmonary circulation. One hour after implant, the heartand lungs are harvested and counted separately. Thrombolysis isexpressed as a percentage where: ##EQU2## The fibrinolytic dissolutionof the implanted clot occurs time-dependently (see J. P. Clozel,Cardiovas. Pharmacol., 12:520, 1988).

Coagulation parameters

Plasma thrombin time (TT) and activated partial thromboplastin time(APTT) are measured with a fibrometer. Blood is sampled from a jugularcatheter and collected in syringe containing sodium citrate (3.8percent, 1 part to 9 parts blood). To measure TT, rat plasma (0.1 mL) ismixed with saline (0.1 mL) and bovine thrombin (0.1 mL, 30 U/mL in TRISbuffer; Parke Davis) at 37° C. For APTT, plasma (0.1 mL) and APTTsolution (0.1 mL, Organon Teknika) are incubated for 5 minutes (37° C.)and CaCl₂ (0.1 mL, 0.025 M) is added to start coagulation. Assays aredone in duplicate and averaged.

Index of Bioavailability

For a measure of bioactivity, plasma thrombin time (TT) serves as asubstitute for the assay of parent compound on the assumption thatobserved increments in TT resulted from thrombin inhibition by parentonly. The time course of the effect of the thrombin inhibitor upon TT isdetermined after i.v bolus administration to anesthetized rats and afteroral treatment of fasted conscious rats. Due to limitations of bloodvolume and the number of points required to determine the time coursefrom time of treatment to the time when the response returns topretreatment values, two populations of rats are used. Each samplepopulation represents alternating sequential time points. The average TTover the time course is used to calculate area under the curve (AUC).The index of bioavailability is calculated by the formula shown belowand is expressed as percent relative activity.

The area under the curve (AUC) of the plasma TT time course isdetermined and adjusted for the dose. This index of bioavailability istermed "% Relative Activity" and is calculated as ##EQU3## Compounds

Compound solutions are prepared fresh daily in normal saline and areinjected as a bolus or are infused starting 15 minutes before andcontinuing throughout the experimental perturbation which is 15 minutesin the arteriovenous shunt model and 60 minutes in the FeCl₃ model ofarterial injury and in the spontaneous thrombolysis model. Bolusinjection volume is 1 mL/kg for i.v., and 5 mL/kg for p.o., and infusionvolume is 3 mL/hr.

Statistics

Results are expressed as means+/-SEM. One-way analysis of variance isused to detect statistically significant differences and then Dunnett'stest is applied to determine which means are different. Significancelevel for rejection of the null hypothesis of equal means is P<0.05.

Animals

Male dogs (Beagles; 18 months-2 years; 12-13 kg, Marshall Farms, NorthRose, N.Y. 14516) are fasted overnight and fed Purina certifiedPrescription Diet (Purina Mills, St. Louis, Mo.) 240 minutes afterdosing. Water is available ad libitum. The room temperature ismaintained between 66-74° F.; 45-50 percent relative humidity; andlighted from 0600-1800 hours.

Pharmacokinetic model

Test compound is formulated immediately prior to dosing by dissolving insterile 0.9 percent saline to a 5 mg/mL preparation. Dogs are given asingle 2 mg/kg dose of test compound by oral gavage. Blood samples (4.5mL) are taken from the cephalic vein at 0.25, 0.5, 0.75, 1, 2, 3, 4 and6 hours after dosing. Samples are collected in citrated Vacutainer tubesand kept on ice prior to reduction to plasma by centrifugation. Plasmasamples are analyzed by HPLC MS. Plasma concentration of test compoundis recorded and used to calculate the pharmacokinetic parameters:elimination rate constant, Ke; total clearance, Clt; volume ofdistribution, V_(D) ; time of maximum plasma test compoundconcentration, Tmax; maximum concentration of test compound of Tmax,Cmax; plasma half-life, t0.5; and area under the curve, A.U.C.; fractionof test compound absorbed, F.

Canine Model of Coronary Artery Thrombosis

Surgical preparation and instrumentation of the dogs are as described inJackson, et al., Circulation, 82, 930-940 (1990). Mixed-breed hounds(aged 6-7 months, either sex, Butler Farms, Clyde, N.Y.) areanesthetized with sodium pentobarbital (30 mg/kg intravenously, i.v.),intubated, and ventilated with room air. Tidal volume and respiratoryrates are adjusted to maintain blood PO₂, PCO₂, and pH within normallimits. Subdermal needle electrodes are inserted for the recording of alead II ECG.

The left jugular vein and common carotid artery are isolated through aleft mediolateral neck incision. Arterial blood pressure (ABP) ismeasured continuously with a precalibrated Millar transducer (model(MPC-500, Millar Instruments, Houston, Tex., U.S.A.) inserted into thecarotid artery. The jugular vein is cannulated for blood sampling duringthe experiment. In addition, the femoral veins of both hindlegs arecannulated for administration of test compound.

A left thoracotomy is performed at the fifth intercostal space, and theheart is suspended in a pericardial cradle. A 1- to 2-cm segment of theleft circumflex coronary artery (LCX) is isolated proximal to the firstmajor diagonal ventricular branch. A 26-gauge needle-tipped wire anodalelectrode (Teflon-coated, 30-gauge silverplated copper wire) 3-4 mm longis inserted into the LCX and placed in contact with the intimal surfaceof the artery (confirmed at the end of the experiment). The stimulatingcircuit is completed by placing the cathode in a subcutaneous (s.c.)site. An adjustable plastic occluder is placed around the LCX, over theregion of the electrode. A precalibrated electromagnetic flow probe(Carolina Medical Electronics, King, N.C., U.S.A.) is placed around theLCX proximal to the anode for measurement of coronary blood flow (CBF).The occluder is adjusted to produce a 40-50 percent inhibition of thehyperemic blood flow response observed after 10-s mechanical occlusionof the LCX. All hemodynamic and ECG measurements are recorded andanalyzed with a data acquisition system (model M3000, ModularInstruments, Malvern, Pa. U.S.A.).

Thrombus Formation and Compound Administration Regimens

Electrolytic injury of the intima of the LCX is produced by applying100-μA direct current (DC) to the anode. The current is maintained for60 min and then discontinued whether the vessel has occluded or not.Thrombus formation proceeds spontaneously until the LCX is totallyoccluded (determined as zero CBF and an increase in the S-T segment).Compound administration is started after the occluding thrombus isallowed to age for 1 hour. A 2-hour infusion of the compounds of thepresent invention at doses of 0.5 and 1 mg/kg/hour is begunsimultaneously with an infusion of thrombolytic agent (e.g. tissueplasminogen activator, streptokinase, APSAC). Reperfusion is followedfor 3 hours after administration of test compound. Reocclusion ofcoronary arteries after successful thrombolysis is defined as zero CBFwhich persisted for at least 30 minutes.

Hematology and template bleeding time determinations

Whole blood cell counts, hemoglobin, and hematocrit values aredetermined on a 40-μL sample of citrated (3.8 percent) blood (1 partcitrate:9 parts blood) with a hematology analyzer (Cell-Dyn 900,Sequoia-Turner. Mount View, Calif., U.S.A.). Gingival template bleedingtimes are determined with a Simplate II bleeding time device (OrganonTeknika Durham, N.C., U.S.A.). The device is used to make two horizontalincisions in the gingiva of either the upper or lower left jaw of thedog. Each incision is 3 mm wide×2 mm deep. The incisions are made, and astopwatch is used to determine how long bleeding occurs. A cotton swabis used to soak up the blood as it oozes from the incision.

Template bleeding time is the time from incision to stoppage ofbleeding. Bleeding times are taken just before administration of testcompound (0 min), 60 min into infusion, at conclusion of administrationof the test compound (120 min), and at the end of the experiment.

All data are analyzed by one-way analysis of variance (ANOVA) followedby Student-Neuman-Kuels post hoc t test to determine the level ofsignificance. Repeated-measures ANOVA are used to determine significantdifferences between time points during the experiments. Values aredetermined to be statistically different at least at the level ofp<0.05. All values are mean±SEM. All studies are conducted in accordancewith the guiding principles of the American Physiological Society.Further details regarding the procedures are described in Jackson, etal., J. Cardiovasc. Pharmacol., (1993), 21, 587-599.

The following Examples are provided to further describe the inventionand are not to be construed as limitations thereof.

The abbreviations, symbols and terms used in the examples have thefollowing meanings.

Ac=acetyl

AIBN=azobisisobutyronitrile

Anal.=elemental analysis

Bn or Bzl=benzyl

Bu=butyl

n-BuLi=butyllithium

Calcd=calculated

DCC=dicyclohexylcarbodiimide

DIBAL-H=diisobutyl aluminum hydride

DMF=dimethylformamide

DMSO=dimethylsulfoxide

Et=ethyl

EtOAc=ethyl acetate

Et₃ N=triethylamine

Et₂ O=diethyl ether

EtOH=ethanol

EtSH=ethanethiol

FAB=Fast Atom Bombardment (Mass Spectroscopy)

FDMS=field desorption mass spectrum

Hex=hexanes

HOAt=1-hydroxy-7-azabenzotriazole

HPLC=High Performance Liquid Chromatography

HRMS=high resolution mass spectrum

i-PrOH=isopropanol

IR=Infrared Spectrum

LAH=lithium aluminum hydride

Me=methyl

MeI=methyl iodide

MeOH=methanol

MPLC=Medium Pressure Liquid Chromatography

NBS=N-bromosuccinimide

NMR=Nuclear Magnetic Resonance

Ph=phenyl

i-Pr=isopropyl

Rochelle's Salt=potassium sodium tartrate

RPHPLC=Reversed Phase High Performance Liquid Chromatography

SiO₂ =silica gel

TBS=tert-butyldimethylsilyl

TFA=trifluoroacetic acid

THF=tetrahydrofuran

TIPS=triisopropylsilyl

TLC=thin layer chromatography

triflic acid=trifluoromethanesulfonic acid

Unless otherwise stated, pH adjustments and work up are with aqueousacid or base solutions. PrepLC indicates preparative liquidchromatography using "Prep Pak(™)" silica cartridges; radialchromatography indicates preparative chromatography using a"Chromatotron(™)" instrument.

EXAMPLE 1 Preparation of6-Hydroxy-2-[4-[2-(1-pyrrolidinyl)ethoxy]-phenyl]-3-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiopheneDioxalate ##STR5## A.6-Isopropoxy-2-[4-(triisopropylsilyloxy)phenyl]-3-bromobenzo[b]thiophene##STR6##

A solution of 15.5 g of the TIPS protected benzothiophene (35.2 mmol) inCH₂ Cl₂ (500 mL) was treated with 6.3 g (35.2 mmol) ofN-bromosuccinimide at ambient temperature for 1 h. The reaction mixturewas concentrated under vacuum and purified by chromatography (SiO₂ ; 1%EtOAc in hexanes) to afford 18 g (34.64 mmol; 98%) of the desiredcompound as a solid.

¹ H NMR (CDCl₃) δ7.69 (d, 1H), 7.61 (d, 2H), 7.26 (s, 1H), 7.05 (m, 1H),6.95 (m, 2H), 4.6 (m, 1H), 1.38 (d, 6H), 1.36-1.30 (m, 3H), 1.18 (d,18H); FDMS 519 (M+); Anal. for C₂₆ H₃₅ BrO₂ SSi: Calcd: C, 60.10; H,6.79; Found: C, 60.37; H, 6.85.

B.6-Isopropoxy-2-[4-(triisopropylsilyloxy)phenyl]-3-bromobenzo[b]thiophene1-Oxide ##STR7##

A solution of the above bromobenzothiophene (9 g, 17.3 mmol)5,10,15,20-tetraphenyl-21H,23H-porphine iron(III) chloride (700 mg, 1mmol) and tetrabutylammonium periodate (15 g, 34.6 mmol) in 500 mL CHCl₃was refluxed for 2 h, concentrated to dryness under vacuum thenredissolved in a mixture of hexanes and CH₂ Cl₂. This solution waspurified by chromatography (SiO₂ ; 5% EtOAc in hexanes) to recover 3.2 gof the starting benzothiophene, 1.1 g of the benzothiophene sulfone and3.6 g (5.9 mmol; 53%) of the desired sulfoxide compound as a solid.

¹ H NMR (CDCl₃) δ7.76-7.73 (m, 2H), 7.55-7.52 (m, 1H), 7.47-7.46 (m,1H), 7.12-7.09 (m, 1H), 7.02-6.99 (m, 2H), 4.7-4.6 (m, 1H), 1.4-1.39 (d,6H), 1.2-1.27 (m, 3H), 1.15-1.13 (d, 18H); FDMS 535 (M+); Anal. for C₂₆H₃₅ BrO₂ SSi.0.25H₂ O: Calcd: C, 57.82; H, 6.62; Found: C, 57.74; H,6.48.

C. 4-[2-(1-Pyrrolidinyl)ethoxy]-1-nitrobenzene ##STR8##

A solution of 10 g of p-nitrophenol (72 mmol) in 100 mL dry THF wastreated with 28.28 g (108 mmol) of triphenyl phosphine. The solution wascooled to 0° C. then treated with 18.78 g (108 mmol) diethylazodicarboxylate. After stirring for 30 min at 0° C., 12.4 g (108 mmol)1-(2-hydroxyethyl)pyrrolidine was added. The cooling bath was removedand the reaction allowed to stir overnight at ambient temperature. EtOAcwas added (300 mL) and the mixture was extracted twice with 200 mL 1 NH₂ SO₄. The combined extracts were washed twice with 200 mL EtOAc, madebasic with 5 N NaOH and extracted three times with 150 mL EtOAc. Theextracts were dried over MgSO₄ and concentrated under vacuum to an oilwhich was purified by chromatography (SiO₂ ; 1% MeOH in CHCl₃) torecover 7.56 g (32 mmol, 44%) of the desired compound as a solid.

¹ H NMR (CDCl₃) δ8.22-7.9 (m, 2H), 7.0-6.9 (m, 2H), 4.22-4.19 (m, 2H),3.0-2.9 (m, 2H), 2.7-2.6 (m, 4H), 1.9-1.8 (m, 4H); FDMS 236 (M+); Anal.for C₂₆ H₃₅ BrO₂ SSi.0.01CHCl₃ : Calcd: C, 60.75; H, 6.80; N, 11.80;Found: C, 60.70; H, 6.66; N, 11.70.

D. 4-[2-(1-Pyrrolidinyl)ethoxy]aniline ##STR9##

The above nitro compound (7.28 g, 30.8 mmol) was mixed with 100 mL MeOH,13 mL 5 N HCl and 1 g 10% Pd/C. This was hydrogenated at atmosphericpressure by bubbling H₂ gas into the stirred solution overnight atambient temperature. The catalyst was filtered off through diatomaceousearth and the filtrate was concentrated under vacuum to dryness yielding8.18 g (29.3 mmol, 93%) of the aniline as its dihydrochloride salt.

¹ H NMR (DMSO-d₆) δ11.4-11.0 (bs, 1H), 10.6-10.2 (bs, 3H), 7.3-7.4 (m,2H), 7.1-7.0 (m, 2H), 4.4-4.3 (m, 2H), 3.6-3.4 (m, 4H), 3.2-3.0 (m, 2H),2.0-1.7 (m, 4H); FDMS 206 (M+): Anal. for C₁₂ H₂₀ Cl₂ N₂ O₂ : Calcd: C,51.62; H, 7.22; N, 10.03; Found: C, 51.72; H, 7.32; N, 9.78.

E. N-[4-[2-(1-Pyrrolidinyl)ethoxy]phenyl]formamide ##STR10##

Acetic anhydride (9.2 g, 90 mmol) was cooled under a nitrogen atmosphereto 0° C. and treated dropwise with formic acid (4.9 g, 108 mmol). Thismixture was stirred at 0° C. for 30 min then warmed to 60° C. for 2 h.The reaction mixture was cooled to room temperature and treated with amixture of the above aniline dihydrochloride (10.1 g, 36 mmol) andtriethylamine (18.3 g, 181 mmol) in 150 mL dry DMF. The reaction wasstirred at ambient temperature for 2 h then concentrated under vacuum toa solid which was mixed with saturated NaHCO₃ and brine. This wasconcentrated to a solid and rinsed with THF. The rinses wereconcentrated to 5.6 g of an oil (24 mmol, 66%).

FDMS 234 (M+); Anal. for C₁₃ H₁₈ N₂ O₂.0.35H₂ O: Calcd: C, 64.90; H,7.83; N, 11.64; Found: C, 64.92; H, 7.60; N, 11.56.

F. N-Methyl-4-[2-(1-pyrrolidinyl)ethoxy]aniline ##STR11##

The above formanilide (4.3 g, 18.4 mmol) was dissolved in dry THF (50mL) and cooled to 0° C. under a nitrogen atmosphere and treated with 19mL of 1M LAH in THF dropwise. This was refluxed for 1 h, cooled to 0° C.and quenched with 200 mL saturated aqueous sodium potassium tartrate.Added 100 mL EtOAc and separated the layers. The aqueous layer wassaturated with NaCl then extracted with 50 mL EtOAc. The combinedorganic layers was dried over MgSO₄ and concentrated to an oil which waspurified by chromatography (SiO₂ ; stepwise 15/83/2 then 19/77/4%THF-Hex-Et₃ N) to recover 2.5 g (11.3 mmol, 62%) of the desired compoundas an oil.

¹ H NMR (DMSO-d₆) δ6.8-6.7 (m, 2H), 6.55-6.45 (m, 2H), 5.2-5.1 (m, 1H),3.95-3.85 (m, 2H), 2.75-2.7 (m, 2H), 2.65-2.57 (m, 3H), 2.55-2.48 (m,4H), 1.7-1.6 (m, 4H); FDMS 221 (M+1); Anal. for C₁₃ H₂₀ N₂ O: Calcd: C,70.87; H, 9.15; N, 12.72; Found: C, 70.79; H, 9.15; N, 12.55.

G.6-Isopropoxy-2-[4-(triisopropylsiloxy)phenyl]-3-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiophene##STR12##

The aniline of Part D, above, (1.5 g, 7.5 mmol) was dissolved in 100 mLdry THF and cooled to -78° C. and treated with 15 mL of 1 M KN[Si(CH₃)₃]₂ in THF. The cooling bath was replaced with an ice bath and themixture was stirred for 1 h before recooling to -78° C. and adding thebenzothiophene sulfoxide of Part B, above, (4 g, 7.5 mmol) as a solid.The reaction was warmed to 0° C. and allowed to stir for 3 h. Brine (200mL) was added followed by EtOAc (100 mL). The layers were separated andthe aqueous layer was extracted twice with 100 mL EtOAc. The combinedorganic layer was dried over MgSO₄ and concentrated to an oil which wasdissolved in 100 mL dry THF and treated with 15 mL 1 M lithiumtri-tert-butoxyaluminum hydride in THF. This was stirred overnight atambient temperature and quenched with 200 mL saturated aqueous sodiumpotassium tartrate and 100 mL EtOAc. After separating, the aqueous layerwas diluted with 200 mL H₂ O and extracted twice with 100 mL EtOAc. Thecombined organic layer was dried over MgSO₄, concentrated under vacuumto an oil and purified by chromatography (SiO₂ ; 20/75/5% THF-Hex-Et₃ N)to recover 2.8 g (4.3 mmol, 58%) of the desired compound as a foam.

¹ H NMR (CDCl₃) δ7.44-7.41 (m, 2H), 7.34-7.32 (m, 1H), 7.25 (s, 1H),6.88-6.85 (m, 3H), 6.80-6.77 (m, 2H), 6.69-6.66 (m, 2H), 5.35 (s, 1H),4.65-4.55 (m, 1H), 4.08-4.00 (m, 2H), 2.9-2.82 (m, 2H), 2.55-2.65 (m,4H), 1.85-1.7 (m, 4H), 1.4-1.35 (m, 6H), 1.3-1.2 (m, 3H), 1.15-1.05 (m,18H); FDMS 645 (M+).

H.6-Isopropoxy-2-(4-hydroxyphenyl)-3-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiophene##STR13##

The above triisopropylsilyl ether (2.7 g, 4.2 mmol) was mixed with 50 mLH₂ O, 50 mL THF and 1 g NaF. This was heated overnight at reflux, cooledto room temperature and extracted with 50 mL EtOAc. The organic layerwas dried over MgSO₄ and concentrated to an oil which crystallized.

¹ H NMR (DMSO-d₆) δ9.63 (s, 1H), 7.45-7.42 (m, 4H), 7.27-7.24 (m, 1H),6.87-6.84 (m, 1H), 6.74-6.71 (m, 2H), 6.66-6.64 (m, 2H), 6.49-6.46 (m,2H), 4.64-4.60 (m, 1H), 3.88-3.84 (m, 2H), 2.69-2.65 (m, 2H), 2.46-2.44(m, 4H), 1.64-1.61 (m, 4H), 1.64-1.62 (m, 6H); FDMS 489 (M+1).

I.6-Isopropoxy-2-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]-3-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiophene##STR14##

A mixture of the above phenol (0.5 g, 1.02 mmol), triphenylphosphine(1.1 g, 4.1 mmol), 1-(2-hydroxyethyl)pyrrolidine (0.47 g, 4.1) and dryTHF (10 mL) was treated with diethyl azodicarboxylate (0.71 g, 4.1mmol). The reaction was allowed to stir overnight at ambienttemperature, concentrated under vacuum and purified by chromatography(SiO₂ ; 40/55/5% THF-Hex-Et₃ N) to recover 490 mg (0.8 mmol, 82%) of thedesired compound as an oil.

¹ H NMR (CDCl₃) δ7.49-7.46 (m, 2H), 7.35-7.32 (m, 1H), 7.25 (s, 1H),6.92-6.85 (m, 3H), 6.80-6.77 (m, 2H), 6.69-6.66 (m, 2H), 5.33 (s, 1H),4.62-4.55 (m, 1H), 4.13-4.03 (m, 4H), 2.92-2.86 (m, 4H), 2.65-2.61 (m,8H), 1.83-1.79 (m, 8H), 1.38-1.36 (m, 6H).

J.6-Hydroxy-2-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]-3-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiopheneDioxalate

The above isopropyl ether (0.49 g, 0.8 mmol) was dissolved in 50 mL CH₂Cl₂ and cooled to 0° C. This was treated with 2 mL 1 M BCl₃ in pentaneat 0° C. for 2 h, concentrated to dryness, and purified bychromatography (SiO₂ ; stepwise 40/55/5 then 80/15/5% THF-Hex-Et₃ N) torecover 148 mg (0.27 mmol, 34%) of the desired compound as a foam.

This was converted to its dioxalate salt by dissolving in MeOH, adding asolution of 2 equivalents of oxalic acid in MeOH and concentrating todryness.

¹ H NMR (DMSO-d₆) δ7.57-7.54 (m, 2H), 7.24-7.20 (m, 2H), 6.97-6.94 (m,2H), 6.79-6.70 (m, 3H), 6.51-6.48 (m, 2H), 5.0 (bs, 6H), 4.23-4.22 (m,2H), 4.09-4.06 (m, 2H), 3.40-3.39 (m, 4H), 3.21-3.19 (m, 8H), 1.95-1.80(m, 8H); FDMS 544 (M+1); Anal. for C₃₂ H₃₇ N₃ O₃ S.2C₂ H₂ O₄ : Calcd: C,59.74; H, 5.71; N, 5.81; Found: C, 59.66; H, 5.70; N, 5.71.

EXAMPLE 2 Preparation of2-[4-[2-(1-Pyrrolidinyl)ethoxy]phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiopheneDioxalate ##STR15## A. 2,3-Dibromobenzo[b]thiophene ##STR16##

Benzothiophene (26.8 g, 0.2 mol) was dissolved in 150 mL CHCl₃ andtreated with a solution of bromine (64 g, 0.4 mol) in 75 mL CHCl₃dropwise over an hour. The reaction was allowed to stir overnight thencautiously quenched with saturated aqueous Na₂ CO₃ until no gasevolution was evident. The layers were separated and the organic layerwas first washed with saturated aqueous Na₂ CO₃ then with water. It wasdried over MgSO₄ and concentrated under vacuum to a solid.Recrystallized from MeOH to obtain 16.5 g (57 mmol, 28%) of a whitefluffy solid.

¹ H NMR (CDCl₃) δ7.77-7.71 (m, 2H), 7.46-7.38 (m, 2H).

B. 2-[4-Methoxyphenyl]-3-bromobenzo[b]thiophene ##STR17##

A mixture of the dibromobenzothiophene (11 g, 37.7 mmol),p-methoxyphenylboronic acid (5.7 g, 37.7 mmol), Pd(OAc) to (58 mg, 0.26mmol) and tri-ortho-tolyl phosphine (237 mg) in 38 mL THF was treatedwith 38 mL 2 M Na₂ CO₃ and refluxed in the absence of light overnight.Diluted with 100 mL EtOAc and separated the layers. The aqueous layerwas extracted with EtOAc (50 mL). The combined organic layer was driedover MgSO₄ and concentrated to an oil which was purified bychromatography (SiO₂ ; stepwise 5 then 8% CHCl₃ in hexanes) to recover9.34 g (29.3 mmol, 78%) of the desired compound as a white solid.

¹ H NMR (DMSO-d₆) δ8.01-7.99 (m, 1H), 7.77-7.74 (m, 1H), 7.66-7.64 (m,2H), 7.53-7.44 (m, 2H), 7.09-7.06 (m, 2H), 3.79 (s, 3H); FDMS 320 (M+1).

C. 2-(4-Hydroxyphenyl)-3-bromobenzo[b]thiophene ##STR18##

The above methyl ether (10 g, 31.35 mmol) was dissolved in 250 mL CH₂Cl₂, cooled to 0° C. and treated with 60 mL 1 M BBr₃ in CH₂ Cl₂. Stirredovernight at ambient temperature. Recooled to 0° C. and quenched with 30mL MeOH. Concentrated to dryness under vacuum, mixed with toluene andreconcentrated to dryness. Purified by chromatography (SiO₂ ; CH₂ Cl₂)to recover 9.4 g (30.8 mmol, 98%) of the desired compound as a whitesolid.

¹ H NMR (DMSO-d₆) δ9.9 (s, 1H), 8.0-7.97 (m, 1H), 7.75-7.73 (m, 1H),7.52-7.4 (m, 2H), 7.52-7.4 (m, 2H), 6.91-6.88 (m, 2H); Anal. for C₁₄ H₉BrSO.0.45H₂ O: Calcd: C, 53.67; H, 3.18; Found: C, 53.67; H, 2.92.

D. 2-[4-(Triisopropylsilyloxy)phenyl]-3-bromobenzo[b]thiophene ##STR19##

A solution of 3-bromo-2-(4-hydroxyphenyl)benzo[b]-thiophene (9.4 g, 30.8mmol) in 50 mL of dry DMF was treated with Et₃ N (6.3 g, 61.6 mmol) andcooled to 0° C. To this was added in a dropwise manner 14.2 g (46.2mmol) triisopropylsilyl trifluoromethanesulfonate. The cooling bath wasremoved and the reaction allowed to stir at ambient temperature for 2 hbefore pouring into 100 mL brine. This was extracted with EtOAc (2×50mL). The combined extracts were washed with brine (2×100 mL), dried overMgSO₄ and concentrated under reduced pressure to 4.8 g of an oil. Thecrude product was purified by chromatography (SiO₂ ; hexanes) to recover11.7 g (25.4 mmol, 82%) of the desired product as a white solid.

¹ H NMR (DMSO-d₆) δ7.85-7.75 (m, 2H), 7.65-7.62 (m, 2H), 7.48-7.35 (m,2H), 7.0-6.95 (m, 2H), 1.37-1.25 (m, 3H), 1.26-1.19 (m, 18H); FDMS 462(M+1); Anal. for C₂₃ H₂₉ BrOSSi: Calcd: C, 59.86; H, 6.33; Found: C,59.58; H, 6.11.

E. 2-[4-(Triisopropylsilyloxy)phenyl]-3-bromobenzo[b]-thiophene 1-Oxide##STR20##

This compound was prepared in 58% yield from the above benzothiophene byessentially following the procedure detailed for the preparation ofExample 1, Part B.

¹ H NMR (CDCl₃) δ7.93-7.91 (m, 1H), 7.79-7.75 (m, 2H), 7.65-7.63 (m,2H), 7.54-7.48 (m, 1H), 7.02-6.99 (m, 2H), 1.38-1.22 (m, 3H), 1.14-1.11(m, 18H); FDMS 479 (M+2); Anal. for C₂₃ H₂₉ BrO₂ SSi: Calcd: C, 57.01;H, 6.19; Found: C, 56.97; H, 5.84.

F.2-[4-(Triisopropylsilyloxy)phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiophene##STR21##

This compound was prepared in 55% yield from the bromide of Example 2,Part E, and the amine of Example 1, Part F, by essentially following theprocedure detailed for the preparation of Example 1, Part G.

¹ H NMR (CDCl₃) δ7.80-7.77 (m, 1H), 7.49-7.46 (m, 2H), 7.31-7.18 (m,4H), 6.87-6.80 (m, 3H), 6.64-6.61 (m, 2H), 4.06-4.04 (m, 2H), 3.15 (s,3H), 2.89-2.85 (m, 2H), 2.61-2.54 (m, 4H), 1.84-1.78 (m, 4H), 1.3-1.2(m, 3H), 1.19-1.08 (m, 18H); FDMS 601 (M+1).

G.2-[4-[2-(1-Pyrrolidinyl)ethoxy]phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiopheneDioxalate

This compound was prepared in 15% yield from Example 2, Part F byessentially following the 2 step procedure detailed for the preparationof Example 1, Part H and Example 1, Part I respectively.

¹ H NMR (CDCl₃) δ7.79-7.78 (m, 1H), 7.56-7.53 (m, 2H), 7.31-7.2 (m, 3H),6.91-6.88 (m, 2H), 6.83-6.79 (m, 2H), 6.64-6.61 (m, 2H), 4.13-4.02 (m,4H), 3.16 (s, 3H), 2.92-2.84 (m, 4H), 2.62-2.53 (m, 8H), 1.81-1.79 (m,8H); FDMS 542 (M+1); Anal. for C₃₃ H₃₉ N₃ SO₂.2C₂ H₂ O₄ : Calcd: C,61.57; H, 6.00; N, 5.82; Found: C, 61.86; H, 6.17; N, 6.04.

EXAMPLE 3 Preparation of6-Hydroxy-2-[4-[2-(1-pyrrolidinyl)ethoxy]-phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]-aminobenzo[b]thiopheneDioxalate ##STR22## A.6-Isopropoxy-2-[4-(triisopropylsilyloxy)phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]-thiophene##STR23##

This compound was prepared in 50% yield from the bromide of Example 1,Part B, and the amine of Example 1, Part F, by essentially following theprocedure detailed for the preparation of Example 1, Part G.

¹ H NMR (CDCl₃) δ7.44-7.42 (m, 2H), 7.26-7.25 (m, 1H), 7.15-7.12 (m,1H), 6.84-6.79 (m, 5H), 6.63-6.60 (m, 2H), 4.62-4.53 (m, 1H), 4.06-4.02(m, 2H), 3.13 (s, 3H), 2.89-2.85 (m, 2H), 2.62-2.54 (m, 4H), 1.82-1.78(m, 4H), 1.37-1.35 (m, 6H), 1.33-1.20 (m, 3H), 1.10-1.07 (m, 18H); FDMS659 (M+).

B.6-Isopropoxy-2-[4-hydroxyphenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiophene##STR24##

This compound was prepared in 26% yield from the silyl ether of Example3, Part A, by essentially following the procedure detailed for thepreparation of Example 1, Part H.

¹ H NMR (CDCl₃) δ7.43-7.40 (m, 2H), 7.234-7.228 (m, 1H), 6.80-6.68 (m,5H), 6.57-6.54 (m, 2H), 5.5 (bs, 1H), 4.60-4.50 (m, 1H), 4.06-4.02 (m,2H), 3.10 (s, 3H), 2.94-2.90 (m, 2H), 2.7-2.65 (m, 4H), 1.83-1.75 (m,4H), 1.34-1.32 (m, 6H); FDMS 503 (M+1).

C.6-Isopropoxy-2-[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiophene##STR25##

A mixture of the above phenol (0.75 g, 1.5 mmol), Cs₂ CO₃ (2 g, 6 mmol),1-(2-chloroethyl)pyrrolidine hydrochloride (0.5 g, 3 mmol) and 20 mL dryDMF was heated at 95° C. for 3 h. It was quenched by pouring into brine(50 mL) and then extracted 3 times with 50 mL 10% MeOH in EtOAc. Theextracts were washed 3 times with 200 mL brine, dried over MgSO₄ andconcentrated to an oil which was purified by chromatography (SiO₂ ; 1%MeOH in CHCl₃ containing NH₄ OH) to recover 280 mg (0.47 mmol, 31%) ofthe desired compound as an oil.

¹ H NMR (CDCl₃) δ7.51-7.48 (m, 2H), 7.26-7.25 (m, 1H), 7.14-7.11 (m,1H), 6.88-6.79 (m, 5H), 6.63-6.6 (m, 2H), 4.59-4.55 (m, 1H), 3.14 (s,3H), 2.92-2.86 (m, 4H), 2.64-2.54 (m, 8H), 1.87-1.80 (m, 8H), 1.36-1.34(m, 6H); FDMS 598 (M-1).

D.2-[4-[2-(1-Pyrrolidinyl)ethoxy]phenyl]-3-(methyl)[4-[2-(1-pyrrolidinyl)ethoxy]phenyl]aminobenzo[b]thiopheneDioxalate

This compound was prepared in 45% yield from the isopropyl ether ofExample 3, Part C, by essentially following the procedure detailed forthe preparation of Example 1, Part J.

¹ H NMR (CDCl₃) δ7.42-7.39 (m, 2H), 7.05-7.02 (m, 2H), 6.73-6.69 (m,5H), 6.56-6.53 (m, 2H), 4.09-4.01 (m, 5H), 3.09 (s, 3H), 2.95-2.87 (m,4H), 2.72-2.64 (m, 8H), 1.84-1.82 (m, 8H); FDMS 558 (M+1); Anal. for C₃₃H₃₉ N₃ O₃ S.1.5C₂ H₂ O₄.1.5 H₂ O: Calcd: C, 62.30; H, 6.57; N, 6.23;Found: C, 62.14; H, 6.61; N, 6.24.

EXAMPLE 4 Preparation of2-[4-[2-(1-Pyrrolidinyl)ethoxy]phenyl]-3-(methyl)[3-methoxy-[4-(1-pyrrolidinyl)phenyl]aminobenzo]-[b]thiopheneDioxalate ##STR26## A. 1-(4-Nitro-2-methoxybenzyl)pyrrolidine ##STR27##

A mixture of 4-nitro-2-methoxytoluene (5 g, 30 mmol), N-bromosuccinimide(5.3 g, 30 mmol), azobisisobutyronitrile (AIBN, 10 mg) and CCl₄ wasrefluxed for 60 h. The reaction mixture was cooled to 0° C., filteredthrough diatomaceous earth and concentrated under vacuum. The resultingoil was redissolved in THF (50 mL) and added portionwise to a cold (0°C.) stirred solution of pyrrolidine (10.65 g, 150 mmol). Afterwards, thecooling bath was removed and the reaction was stirred at ambienttemperature for 1 h. It was then concentrated to an oil, diluted with100 mL saturated aqueous NaHCO₃ and 50 mL brine. Following extractionthree times with 125 mL EtOAc, the combined organic layer was dried overMgSO₄ and concentrated to 6.5 g of an oil. Purification bychromatography (SiO₂ ; 30% EtOAc in hexanes) yielded 5.7 g (24 mmol,80%) of the desired compound as an oil.

¹ H NMR (CDCl₃) δ7.86-7.83 (m, 1H), 7.71-7.7.0 (m, 1H), 7.59-7.56 (m,1H), 3.94 (s, 3H), 3.72 (s, 2H), 2.61-2.57 (m, 4H), 1.85-1.81 (m, 4H);FDMS 236 (M+).

B. 4-(1-Pyrrolidinylmethyl)-3-methoxyaniline ##STR28##

The above nitro compound (5.5 g, 23 mmol) was dissolved in 50 mL MeOHwith 0.6 g 10% Pd/C. This was hydrogenated at atmospheric pressure bybubbling H₂ gas into the mixture for 6 h after which the catalyst wasremoved by filtration through diatomaceous earth. The filtrate wasconcentrated under vacuum to afford 4.7 g of an oil (22.8 mmol, 99%).

¹ H NMR (CDCl₃) δ7.2-7.17 (m, 1H), 6.30-6.27 (m, 1H), 6.24-6.23 (m, 1H),3.81 (s, 2H), 3.80 (s, 3H), 2.85-2.80 (m, 4H), 1.92-1.87 (m, 4H); FDMS207 (M+1).

C. N-[4-(1-Pyrrolidinylmethyl)-3-methoxyphenyl]formamide ##STR29##

The above aniline (5.5 g, 26.7 mmol) was mixed with ethyl formate (7.9g, 107 mmol) and heated overnight at 110° C. Excess ethyl formate wasremoved under reduced pressure and the residue was purified bychromatography (SiO₂ ; stepwise 1 then 5% MeOH in CHCl₃) to give 4.92 g(21 mmol, 78%) of the desired compound as an oil.

¹ H NMR was complicated by the existence of rotamers. Characteristic NMRof this compound is as follows: (CDCl₃) 8.6-8.7 (m, 1/2H), 8.35-8.38 (m,1/2H), 7.7 (bs, 1/2H), 7.5 (bs, 1/2H), 7.45 (m, 1/2H), 7.3-7.23 (m, 2H),6.82-6.80 (m, 1/2H), 6.5-6.61 (m, 1/2H), 6.5-6.43 (m, 1/2H), 3.8 (s,3H), 3.6 (s, 2H), 2.6-2.5 (m, 4H), 1.8-1.7 (m, 4H); IR (CHCl₃ 2966.84,1695.44, 1612.21, 1514.29, 1463.97, 1415.96 cm¹); FDMS 235 (M+1); Anal.for C₁₃ H₁₈ N₂ O₂.0.11CHCl₃ : Calcd: C, 63.64; H, 7.38; N, 7.38; Found:C, 63.71; H, 7.04; N, 11.41.

D. 4-(1-Pyrrolidinylmethyl)-N-methyl-3-methoxyaniline ##STR30##

This compound was prepared in 71% yield from the above formula byessentially following the procedure detailed for the preparation ofExample 1, Part F.

¹ H NMR (CDCl₃) δ7.11-7.08 (m, 1H), 6.20-6.17 (m, 1H), 6.15-6.14 (m,1H), 3.79 (s, 3H), 3.65 (bs, 1H), 3.56 (s, 2H), 3.84-2.83 (m, 3H),2.58-2.51 (m, 4H), 1.80-1.73 (m, 4H); FDMS 220 (M+).

E.2-[4-(Triisopropylsilyloxy)phenyl]-3-(methyl)-[3-methoxy-[4-(1-pyrrolidinylmethyl]phenyl]aminobenzo[b]thiophene##STR31##

This compound was prepared in 77% yield from the amine of Example 4,Part D, and the bromide of Example 2, Part E, by essentially followingthe procedure detailed for the preparation of Example 1, Part G.

¹ H NMR (CDCl₃) δ7.80-7.72 (m, 1H), 7.47-7.45 (m, 2H), 7.33-7.20 (m,3H), 7.11-7.09 (m, 1H), 6.87-6.83 (m, 2H), 6.24-6.27 (m, 1H), 6.18-6.17(m, 1H), 3.16 (s, 3H), 3.56 (s, 2H), 3.17 (s, 3H), 2.6-2.53 (m, 4H),1.77-1.67 (m, 4H), 1.23-1.29 (m, 3H), 1.18-1.02 (m, 18H); FDMS 601(M+1); Anal. for C₃₆ H₄₈ N₂ O₂ SSi: Calcd: C, 71.95; H, 8.05; N, 4.66;Found: C, 72.12; H, 7.89; N, 4.66.

F.2-(4-Hydroxyphenyl)-3-(methyl)[3-methoxy-[4-[(1-pyrrolidinyl)methyl]phenylamino]]benzo[b]thiophene##STR32##

This compound was prepared in 96% yield from the silyl ether of Example4, Part E, by essentially following the procedure detailed for thepreparation of Example 1, Part H.

¹ H NMR (CDCl₃) δ7.77-7.45 (m, 1H), 7.41-7.38 (m, 2H), 7.29-7.20 (m,3H), 7.12-7.09 (m, 1H), 6.74-6.71 (m, 2H), 6.23-6.20 (m, 1H), 6.11 (s,1H), 3.68 (s, 2H), 3.47 (s, 3H), 3.15 (s, 3H), 2.95 (bs, 1H), 2.73-2.71(m, 4H), 1.83-1.81 (m, 4H); FDMS 445 (M+1).

G.2-[4-[2-(1-Pyrrolidinyl)ethoxy]phenyl]-3-(methyl)-[3-methoxy-[4-(1-pyrrolidinyl)phenyl]aminobenzo][b]thiopheneDioxalate

This compound was prepared in 81% yield from the phenol of Example 4,Part F, by essentially following the procedure detailed for thepreparation of Example 1, Part I.

¹ H NMR (CDCl₃, Free Base) δ7.80-7.77 (m, 1H), 7.54-7.51 (m, 2H),7.33-7.22 (m, 3H), 7.13-7.10 (m, 1H), 6.91-6.88 (m, 2H), 6.28-6.24 (m,1H), 6.18-6.17 (m, 1H), 4.13-4.09 (m, 2H), 3.62 (s, 3H), 3.58 (s, 2H),3.19 (s, 3H), 2.92-2.88 (m, 2H), 2.67-2.57 (m, 8H), 1.82-1.77 (m, 8H);FDMS 542 (M+1); Anal. for C₃₃ H₃₉ N₃ O₂ S.2.62C₂ H₂ O₄ : Calcd: C,59.06; H, 5.73; N, 5.40; Found: C, 59.08; H, 5.62; N, 5.44.

EXAMPLE 5 Preparation of2-[4-(4-Methoxy-4-oxobutyloxy)phenyl]-3-(methyl)[3-methoxy-[4-(1-pyrrolidinylmethyl)phenyl]aminobenzo][b]thiopheneOxalate ##STR33##

A mixture of the phenol of Example 4, Part F (0.75 g, 1.7 mmol), Cs₂ CO₃(1.1 g, 3.4 mmol), methyl 4-chlorobutyrate (0.28 g, 2 mmol) and 10 mLdry DMF was heated at 90° C. for 3 h. The reaction was filtered whilehot, then concentrated under reduced pressure to an oil. The residue waspurified by chromatography (SiO₂ ; 20/75/5% THF/Hex/Et₃ N) to recover797 mg (1.6 mmol, 86%) of the desired compound as an oil. This wasconverted to its oxalate salt by dissolving in EtOAc, adding a solutionof 1 equivalent of oxalic acid in EtOAc and concentrating to a foamunder reduced pressure.

¹ H NMR (CDCl₃ -- Free Base) δ7.80-7.77 (m, 1H), 7.54-7.53 (m, 2H),7.51-7.20 (m, 3H), 7.12-7.09 (m, 1H), 6.88-6.84 (m, 2H), 6.27-6.24 (m,1H), 6.18-6.17 (m, 1H), 4.03-3.99 (m, 2H), 3.69-3.68 (m, 3H), 3.62 (s,3H), 3.18-317 (m, 3H), 2.55-2.49 (m, 4H), 2.16-2.07 (m, 2H), 1.79-1.69(m, 4H); FDMS 545 (M+1); Anal. for C₃₂ H₃₆ N₂ O₄ S.1.11C₂ H₂ O₄ : Calcd:C, 63.76, H, 5.98; N, 4.35; Found: C, 63.78; H, 5.83; N, 4.20.

EXAMPLE 6 Preparation of2-[4-[3-(Carboxy)propoxy]phenyl]-3-(methyl)-[3-methoxy-[4-(1-pyrrolidinylmethyl)phenyl]aminobenzo][b]thiopheneOxalate ##STR34##

The ester of Example 5 (0.39 g, 0.72 mmol) was dissolved in 30 mL MeOHand treated with 2.1 mL 1 N NaOH. The reaction was warmed to 50° C. for16 h then neutralized with 1 N HCl and concentrated to dryness underreduced pressure. The residue was dissolved 10% MeOH in CHCl₃ andpurified by chromatography (SiO₂ ; stepwise 10, 20, then 30% CHCl₃ inMeOH) to recover 175 mg (0.33 mmol, 46%) of the desired compound as afoam. This was converted to its oxalate salt by dissolving in MeOH,adding a solution of 1 eq oxalic acid in MeOH and concentrating todryness under reduced pressure.

¹ H NMR (DMSO-d₆ Free Base) δ8.95-8.90 (m, 1H), 7.48-7.45 (m, 2H),7.39-7.20 (m, 3H), 7.15-7.10 (m, 1H), 6.98-6.92 (m, 2H), 6.3 (s, 1H),6.08-6.18 (m, 1H), 4.05-3.95 (m, 4H), 3.67 (s, 3H), 3.60-3.55 (m, 2H),3.23 (s, 3H), 3.18-2.98 (m, 4H), 2.40-2.35 (m, 2H), 1.92-1.8 (m, 4H);FDMS 531 (M+1).

What is claimed is:
 1. A compound of formula I or a pharmaceuticallyacceptable salt thereof ##STR35## wherein E is CR^(e) or N in whichR^(e) is hydrogen, methyl, methoxy or halo;R¹ is hydrogen or methyl; R²is R^(2a) or R^(2b) in whichR^(2a) is --X² --(CH₂)_(n) --R^(f) in whichX² is a direct bond, methylene or O; n is 1, 2 or 3; and R^(f) is5-tetrazolyl, carboxy, [(1-4C)alkoxy]carbonyl or hydroxymethyl; R^(2b)is --X² --(CH₂)_(m) --NR^(a) R^(b) in which X² is a direct bond,methylene or O; m is 1, 2 or 3; provided that when m is 1, then X² is adirect bond; and R^(a) and R^(b) are independently hydrogen or(1-3C)alkyl or the group NR^(a) R^(b) is pyrrolidino, piperidino ormorpholino; R³ is --X³ --(CH₂)_(s) --NR^(s) R^(t) in which X³ is adirect bond, methylene or O; s is 1 or 2; provided that when s is 1,then X³ is a direct bond; and R^(s) and R^(t) are independently hydrogenor (1-3C)alkyl or the group NR^(s) R^(t) is pyrrolidino, piperidino ormorpholino; and R⁶ is hydrogen, hydroxy or methoxy.
 2. The compound ofclaim 1 wherein (1-3C)alkyl is methyl, ethyl, propyl or isopropyl;(1-4C)alkoxy is methoxy, ethoxy, propoxy, isopropoxy, butoxy ortert-butoxy; and halo is fluoro, chloro, bromo or iodo.
 3. The compoundof claim 1 or 2 wherein E is CR^(e) in which R^(e) is hydrogen ormethoxy;R¹ is hydrogen or methyl; R² is R² a or R^(2b) in whichR^(2a) is--X² --(CH₂)_(n) --R^(f) in which X² is O; n is 2; and R^(f) is carboxyor methoxycarbonyl; R^(2b) is --X² --(CH₂)_(m) --NR^(a) R^(b) in whichX² is O; m is 2; and the group NR^(a) R^(b) is pyrrolidino; R³ is --X³--(CH₂)_(s) --NR^(s) R^(t) in which X³ is a direct bond or O; s is 1 or2; provided that when s is 1, then X³ is a direct bond; and the groupNR^(s) R^(t) is pyrrolidino; and R⁶ is hydrogen or hydroxy.
 4. Thecompound of claim 2 wherein R² is R^(2a).
 5. The compound of claim 2wherein R² is R^(2b).
 6. The compound as claimed in claim 3 wherein E isCR^(e) in which R^(e) methoxy and R³ is pyrrolidinomethyl.
 7. Thecompound as claimed in claim 4 wherein E is CR^(e) in which R^(e)methoxy and R³ is pyrrolidinomethyl.
 8. The compound as claimed in claim5 wherein E is CR^(e) in which R^(e) methoxy and R³ ispyrrolidinomethyl.
 9. The compound of claim 1 which is2-[4-[3-(carboxy)propoxy]phenyl]-3-(methyl)[3-methoxy-[4-(1-pyrrolidinylmethyl)phenyl]aminobenzo][b]thiophene.10. A pharmaceutically acceptable salt of a compound of formula I asclaimed in claim 1 which is an acid-addition salt made with an acidwhich provides a pharmaceutically acceptable anion or which is a saltmade with a base which provides a pharmaceutically acceptable anion. 11.A pharmaceutical formulation comprising in association with apharmaceutically acceptable carrier, diluent or excipient, a compound offormula I as provided in claim
 1. 12. A process for preparing a compoundof formula I as claimed in claim 1 which is selected from:(a) for acompound of formula I in which R² is --X² --(CH₂)_(n) --R^(f) or --X²--(CH₂)_(m) NR^(a) R^(b) where X² is O, alkylating a correspondingphenol of formula II ##STR36## using a compound of formula L-(CH₂)_(n)--R^(f) or L-(CH₂)_(m) --NR^(a) R^(b) wherein L denotes a conventionalleaving group using a standard alkylating procedure; (b) for a compoundof formula I in which R^(f) is carboxy, decomposing the ester of acorresponding compound of formula I in which R^(f) is {(14C)alkoxy}carbonyl; and (c) deoxygenating the sulfoxide of acorresponding compound of formula III ##STR37## whereafter, for any ofthe above procedures, when a functional group is protected using aprotected group, removing the protecting group; and whereafter, for anyof the above procedures, when a pharmaceutically acceptable salt of acompound of formula I is required, it is obtained by reacting the basicform of such a compound of formula I with an acid affording aphysiologically acceptable counterion, or, for a compound of formula Iwhich bears an acidic moiety, reacting the acidic form of such acompound of formula I with a base which affords a pharmaceuticallyacceptable cation, or by any other conventional procedure; and wherein,unless otherwise described, R¹, R², R³ and R⁶ have the values describedin claim
 1. 13. A method of inhibiting thrombin comprising administeringan effective amount of a compound of formula I as described in claim 1.